Monique M. van Oers

Opportunities and challenges for the baculovirus expression system

In this review background information on the baculovirus-insect cell expression system and its applications for producing protein subunits and virus-like particles for vaccine and other purposes is provided. This review will illustrate the principle structure of baculovirus vectors commonly used for heterologous gene expression in insect cells and describe adaptations that have been made over the last 10 years to improve the system in terms of quality of the protein produced and stability of the baculovirus genome. These improvements include enhanced trafficking, folding and glycosylation of the recombinant protein as well as preventing intracellular degradation. Challenges and progress in stabilizing the baculovirus genome in order not to lose the transgene cassette will also be discussed. Recent developments such as how to make multiple alterations in the baculovirus genome without accumulating marker genes are included.

Baculovirus Per Os Infectivity Factors Form a Complex on the Surface of Occlusion-Derived Virus

ABSTRACT Five highly conserved per os infectivity factors, PIF1, PIF2, PIF3, PIF4, and P74, have been reported to be essential for oral infectivity of baculovirus occlusion-derived virus (ODV) in insect larvae. Three of these proteins, P74, PIF1, and PIF2, were thought to function in virus binding to insect midgut cells. In this paper evidence is provided that PIF1, PIF2, and PIF3 form a stable complex on the surface of ODV particles of the baculovirus Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV). The complex could withstand 2% SDS-5% β-mercaptoethanol with heating at 50°C for 5 min. The complex was not formed when any of the genes for PIF1, PIF2, or PIF3 was deleted, while reinsertion of these genes into AcMNPV restored the complex. Coimmunoprecipitation analysis independently confirmed the interactions of the three PIF proteins and revealed in addition that P74 is also associated with this complex. However, deletion of the p74 gene did not affect formation of the PIF1-PIF2-PIF3 complex. Electron microscopy analysis showed that PIF1 and PIF2 are localized on the surface of the ODV with a scattered distribution. This distribution did not change for PIF1 or PIF2 when the gene for PIF2 or PIF1 protein was deleted. We propose that PIF1, PIF2, PIF3, and P74 form an evolutionarily conserved complex on the ODV surface, which has an essential function in the initial stages of baculovirus oral infection.

Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells

BackgroundChikungunya virus (CHIKV) is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively.ResultsExpression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf 21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits.ConclusionsChikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections.

Walking with insects: molecular mechanisms behind parasitic manipulation of host behaviour

Parasitic infections are often followed by changes in host behaviour. Numerous and exquisite examples of such behavioural alterations are known, covering a broad spectrum of parasites and hosts. Most descriptions of such parasite‐induced changes in host behaviour are observational reports, while experimentally confirmed examples of parasite genes inducing these changes are limited. In this study, we review changes in invertebrate host behaviour observed upon infection by parasites and discuss such changes in an evolutionary context. We then explore possible mechanisms involved in parasite‐induced changes in host behaviour. Genes and pathways known to play a role in invertebrate behaviour are reviewed, and we hypothesize how parasites (may) affect these pathways. This review provides the state of the art in this exciting, interdisciplinary field by exploring possible pathways triggered in hosts, suggesting methodologies to unravel the molecular mechanisms that lead to changes in host behaviour.

Characterization of Novel Components of the Baculovirus Per Os Infectivity Factor Complex

ABSTRACT Baculovirus occlusion-derived virus (ODV) infects insect midgut cells under alkaline conditions, a process mediated by highly conserved per os infectivity factors (PIFs), P74 (PIF0), PIF1, PIF2, PIF3, PIF4, and PIF5 (ODV-E56). Previously, a multimolecular complex composed of PIF1, PIF2, PIF3, and P74 was identified which was proposed to play an essential role during ODV entry. Recently, more proteins have been identified that play important roles in ODV oral infectivity, including PIF4, PIF5, and SF58, which might work in concert with previously known PIFs to facilitate ODV infection. In order to understand the ODV entry mechanism, the identification of all components of the PIF complex is crucial. Hence, the aim of this study was to identify additional components of the PIF complex. Coimmunoprecipitation (CoIP) combined with proteomic analysis was used to identify the components of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) PIF complex. PIF4 and P95 (AC83) were identified as components of the PIF complex while PIF5 was not, and this was confirmed with blue native PAGE and a second CoIP. Deletion of the pif4 gene impaired complex formation, but deletion of pif5 did not. Differentially denaturing SDS-PAGE further revealed that PIF4 forms a stable complex with PIF1, PIF2, and PIF3. P95 and P74 are more loosely associated with this complex. Three other proteins, AC5, AC68, and AC108 (homologue of SF58), were also found by the proteomic analysis to be associated with the PIF complex. Finally the functional significance of the PIF protein interactions is discussed.

Engineering of baculovirus vectors for the manufacture of virion-free biopharmaceuticals.

A novel baculovirus‐based protein expression strategy was developed to produce recombinant proteins in insect cells without contaminating baculovirus virions. This novel strategy greatly simplifies the downstream processing of biopharmaceuticals produced in insect cells. The formation of these virions is prevented by deletion of a baculovirus gene essential for virion formation. The deletion is trans‐complemented in a transgenic insect cell line in which the baculovirus seed stock is produced. The Autographa californica multicapsid nucleopolyhedrovirus vp80 gene was selected for this purpose, as absence of VP80 prevented the formation of budded virus as well as occlusion‐derived virus, while foreign gene expression was not affected. Sf9 insect cells were engineered to functionally complement the vp80 deletion in the expression vector virus during seed stock production. The trans‐complemented vp80‐deletion baculovirus seed produced an amount of recombinant protein similar to that produced with conventional baculovirus vectors but without contaminating virions. This novel expression method obviates the need to purify the virions away from the biopharmaceuticals. Bioeng. 2011; 108:1056–1067.

Protein tyrosine phosphatase-induced hyperactivity is a conserved strategy of a subset of baculoviruses to manipulate lepidopteran host behavior.

Many parasites manipulate host behavior to increase the probability of transmission. To date, direct evidence for parasitic genes underlying such behavioral manipulations is scarce. Here we show that the baculovirus Autographa californica nuclear polyhedrovirus (AcMNPV) induces hyperactive behavior in Spodoptera exigua larvae at three days after infection. Furthermore, we identify the viral protein tyrosine phosphatase (ptp) gene as a key player in the induction of hyperactivity in larvae, and show that mutating the catalytic site of the encoded phosphatase enzyme prevents this induced behavior. Phylogenetic inference points at a lepidopteran origin of the ptp gene and shows that this gene is well-conserved in a group of related baculoviruses. Our study suggests that ptp-induced behavioral manipulation is an evolutionarily conserved strategy of this group of baculoviruses to enhance virus transmission, and represents an example of the extended phenotype concept. Overall, these data provide a firm base for a deeper understanding of the mechanisms behind baculovirus-induced insect behavior.

Baculovirus VP80 Protein and the F-Actin Cytoskeleton Interact and Connect the Viral Replication Factory with the Nuclear Periphery

ABSTRACT Recently, we showed that the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) VP80 protein is essential for the formation of both virion types, budded virus (BV) and occlusion-derived virus (ODV). Deletion of the vp80 gene did not affect assembly of nucleocapsids. However, these nucleocapsids were not able to migrate from the virogenic stroma to the nuclear periphery. In the current paper, we constructed a baculovirus recombinant with enhanced-green fluorescent protein (EGFP)-tagged VP80, allowing visualization of the VP80 distribution pattern during infection. In baculovirus-infected cells, the EGFP-VP80 protein is entirely localized in nuclei, adjacent to the virus-triggered F-actin scaffold that forms a highly organized three-dimensional network connecting the virogenic stroma physically with the nuclear envelope. Interaction between VP80 and host actin was confirmed by coimmunoprecipitation. We further showed that VP80 is associated with the nucleocapsid fraction of both BVs and ODVs, typically at one end of the nucleocapsids. In addition, the presence of sequence motifs with homology to invertebrate paramyosin proteins strongly supports a role for VP80 in the polar transport of nucleocapsids to the periphery of the nucleus on their way to the plasma membrane to form BVs and for assembly in the nuclear periphery to form ODVs for embedding in viral occlusion bodies.

Specificity of Baculovirus P6.9 Basic DNA-Binding Proteins and Critical Role of the C Terminus in Virion Formation†

ABSTRACT The majority of double-stranded DNA (dsDNA) viruses infecting eukaryotic organisms use host- or virus-expressed histones or protamine-like proteins to condense their genomes. In contrast, members of the Baculoviridae family use a protamine-like protein named P6.9. The dephosphorylated form of P6.9 binds to DNA in a non-sequence-specific manner. By using a p6.9-null mutant of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), we demonstrate that P6.9 is not required for viral DNA replication but is essential for the production of infectious virus. Virion production was rescued by P6.9 homologs from a number of Alphabaculovirus species and one Gammabaculovirus species but not from the genus Betabaculovirus, comprising the granuloviruses, or by the P6.9 homolog VP15 from the unrelated white spot syndrome virus of shrimp. Mutational analyses demonstrated that AcMNPV P6.9 with a conserved 11-residue deletion of the C terminus was not capable of rescuing p6.9-null AcMNPV, while a chimeric Betabaculovirus P6.9 containing the P6.9 C-terminal region of an Alphabaculovirus strain was able to do so. This implies that the C terminus of baculovirus P6.9 contains sequence elements essential for virion formation. Such elements may possibly interact with species- or genus-specific domains of other nucleocapsid proteins during virus assembly.

Encyclopedia of Autographa californica nucleopolyhedrovirus genes

The Autographa californica multiple capsid nucleopolyhedrovirus (AcMNPV) was the first baculovirus for which the complete nucleotide sequence became known. Since then 15 years lapsed and much research has been performed to elucidate putative functions of the annotated open reading frames of this virus and this endeavour is still ongoing. AcMNPV is the most well-known and well-studied baculovirus species, not in the least for its application as a vector for the high-level expression of foreign genes in insect cells. This article is the first monograph of a single baculovirus and gives a current overview of what is known about the 151 AcMNPV ORFs, including (putative) function and temporal and spatial presence of transcripts and protein. To date 60 ORFs have a proven function, another 19 ORFs have homologs for which functions are known in other baculoviruses and 72 ORFs are still enigmatic. This paper should assist the reader in quickly finding the essentials of AcMNPV.